Intravascular delivery of non-viral nucleic acid

ABSTRACT

Disclosed is a complex for providing nucleic acid expression in a cell. A polynucleotide and a polymer are mixed together to form the complex wherein the zeta potential of the complex is not positive. Then the complex is delivered to the cell wherein the nucleic acid is expressed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] (Provisional Application Serial No.) (Filing Date) 60/121,730Feb. 26, 1999 60/146,564 Jul. 30, 1999

FEDERALLY SPONSORED RESEARCH

[0002] N/A

[0003] 1. Field of the Invention

[0004] The invention relates to compounds and methods for use inbiologic systems. More particularly, processes that transfer nucleicacids into cells are provided. Nucleic acids in the form of naked DNA ora nucleic acid combined with another compound are delivered to cells.

[0005] 2. Background

[0006] Biotechnology includes the delivery of a genetic information to acell to express an exogenous nucleotide sequence, to inhibit, eliminate,augment, or alter expression of an endogenous nucleotide sequence, or toexpress a specific physiological characteristic not naturally associatedwith the cell. Polynucleotides may be coded to express a whole orpartial protein, or may be anti-sense.

[0007] A basic challenge for biotechnology and thus its subpart, genetherapy, is to develop approaches for delivering genetic information tocells of a patient in a way that is efficient and safe. This problem of“drug delivery,” where the genetic material is a drug, is particularlychallenging. If genetic material are appropriately delivered they canpotentially enhance a patient's health and, in some instances, lead to acure. Therefore, a primary focus of gene therapy is based on strategiesfor delivering genetic material in the form of nucleic acids. Afterdelivery strategies are developed they may be sold commercially sincethey are then useful for developing drugs.

[0008] Delivery of a nucleic acid means to transfer a nucleic acid froma container outside a mammal to near or within the outer cell membraneof a cell in the mammal. The term transfection is used herein, ingeneral, as a substitute for the term delivery, or, more specifically,the transfer of a nucleic acid from directly outside a cell membrane towithin the cell membrane. The transferred (or transfected) nucleic acidmay contain an expression cassette. If the nucleic acid is a primary RNAtranscript that is processed into messenger RNA, a ribosome translatesthe messenger RNA to produce a protein within the cytoplasm. If thenucleic acid is a DNA, it enters the nucleus where it is transcribedinto a messenger RNA that is transported into the cytoplasm where it istranslated into a protein. Therefore if a nucleic acid expresses itscognate protein, then it must have entered a cell. A protein maysubsequently be degraded into peptides, which may be presented to theimmune system.

[0009] It was first observed that the in vivo injection of plasmid DNAinto muscle enabled the expression of foreign genes in the muscle(Wolff, J A, Malone, R W, Williams, P, et al. Direct gene transfer intomouse muscle in vivo. Science 1990;247:1465-1468.). Since that report,several other studies have reported the ability for foreign geneexpression following the direct injection of DNA into the parenchyma ofother tissues. Naked DNA was expressed following its injection intocardiac muscle (Acsadi, G., Jiao, S., Jani, A., Duke, D., Williams, P.,Chong, W., Wolff, J. A. Direct gene transfer and expression into ratheart in vivo. The New Biologist 3(1), 71-81, 1991.).

SUMMARY

[0010] In one preferred embodiment, a process is described fordelivering a polynucleotide into a parenchymal cell of a mammal,comprising making a polynucleotide such as a nucleic acid. Then,inserting the polynucleotide into a mammalian vessel, such as a bloodvessel and increasing the permeability of the vessel. Finally,delivering the polynucleotide to the parenchymal cell thereby alteringendogenous properties of the cell. Increasing the permeability of thevessel consists of increasing pressure against vessel walls. Increasingthe pressure consists of increasing a volume of fluid within the vessel.Increasing the volume consists of inserting the polynucleotide in asolution into the vessel wherein the solution contains a compound whichcomplexes with the polynucleotide. A specific volume of the solution isinserted within a specific time period. Increased pressure is controlledby altering the specific volume of the solution in relation to thespecific time period of insertion. The vessel may consist of a tailvein. The parenchymal cell is a cell selected from the group consistingof liver cells, spleen cells, heart cells, kidney cells and lung cells.

[0011] In another embodiment, a complex for providing nucleic acidexpression in a cell is provided, comprising mixing a polynucleotide anda polymer to form the complex wherein the zeta potential of the complexis not positive. Then, delivering the complex to the cell wherein thenucleic acid is expressed.

[0012] In another embodiment, a process is described for delivering apolynucleotide complexed with a compound into a parenchymal cell of amammal, comprising making the polynucleotide-compound complex whereinthe compound is selected from the group consisting of amphipathiccompounds, polymers and non-viral vectors. Inserting the polynucleotideinto a mammalian vessel and increasing the permeability of the vessel.Then, delivering the polynucleotide to the parenchymal cell therebyaltering endogenous properties of the cell.

[0013] In yet another embodiment, a process is described fortransfecting genetic material into a mammalian cell, comprisingdesigning the genetic material for transfection. Inserting the geneticmaterial into a mammalian blood vessel. Increasing permeability of theblood vessel and delivering the genetic material to the parenchymal cellfor the purpose of altering endogenous properties of the cell.

DETAILED DESCRIPTION

[0014] We have found that an intravascular route of administrationallows a polynucleotide to be delivered to a parenchymal cell in a moreeven distribution than direct parenchymal injections. The efficiency ofpolynucleotide delivery and expression is increased by increasing thepermeability of the tissue's blood vessel. Permeability is increased byincreasing the intravascular hydrostatic (physical) pressure, deliveringthe injection fluid rapidly (injecting the injection fluid rapidly),using a large injection volume, and increasing permeability of thevessel wall. Expression of a foreign DNA is obtained in large number ofmammalian organs including; liver, spleen, lung, kidney and heart byinjecting the naked polynucleotide. Increased expression occurs whenpolynucleotide is mixed with another compound.

[0015] In a first embodiment the compound consists of an amphipathiccompound. Amphipathic compounds have both hydrophilic (water-soluble)and hydrophobic (water-insoluble) parts. The amphipathic compound can becationic or incorporated into a liposome that is positively-charged(cationic) or non-cationic which means neutral, or negatively-charged(anionic). In another embodiment the compound consists of a polymer. Inyet another embodiment the compound consists of a non-viral vector. Inone embodiment, the compound does not aid the transfection process invitro of cells in culture but does aid the delivery process in vivo inthe whole organism. We also show that foreign gene expression can beachieved in hepatocytes following the rapid injection of naked plasmidDNA in a large volume of physiologic solutions.

[0016] The term intravascular refers to an intravascular route ofadministration that enables a polymer, oligonucleotide, orpolynucleotide to be delivered to cells more evenly distributed thandirect injections. Intravascular herein means within an internal tubularstructure called a vessel that is connected to a tissue or organ withinthe body of an animal, including mammals. Within the cavity of thetubular structure, a bodily fluid flows to or from the body part.Examples of bodily fluid include blood, lymphatic fluid, or bile.Examples of vessels include arteries, arterioles, capillaries, venules,sinusoids, veins, lymphatics, and bile ducts. The intravascular routeincludes delivery through the blood vessels such as an artery or a vein.

[0017] Afferent blood vessels of organs are defined as vessels in whichblood flows toward the organ or tissue under normal physiologicconditions. Efferent blood vessels are defined as vessels in which bloodflows away from the organ or tissue under normal physiologic conditions.In the heart, afferent vessels are known as coronary arteries, whileefferent vessels are referred to as coronary veins.

[0018] The term naked nucleic acids indicates that the nucleic acids arenot associated with a transfection reagent or other delivery vehiclethat is required for the nucleic acid to be delivered to a target cell.A transfection reagent is a compound or compounds used in the prior artthat mediates nucleic acids entry into cells.

[0019] Parenchymal Cells

[0020] Parenchymal cells are the distinguishing cells of a gland ororgan contained in and supported by the connective tissue framework. Theparenchymal cells typically perform a function that is unique to theparticular organ. The term “parenchymal” often excludes cells that arecommon to many organs and tissues such as fibroblasts and endothelialcells within blood vessels.

[0021] In a liver organ, the parenchymal cells include hepatocytes,Kupffer cells and the epithelial cells that line the biliary tract andbile ductules. The major constituent of the liver parenchyma arepolyhedral hepatocytes (also known as hepatic cells) that presents atleast one side to an hepatic sinusoid and opposed sides to a bilecanaliculus. Liver cells that are not parenchymal cells include cellswithin the blood vessels such as the endothelial cells or fibroblastcells. In one preferred embodiment hepatocytes are targeted by injectingthe polynucleotide within the tail vein of a rodent such as a mouse.

[0022] In striated muscle, the parenchymal cells include myoblasts,satellite cells, myotubules, and myofibers. In cardiac muscle, theparenchymal cells include the myocardium also known as cardiac musclefibers or cardiac muscle cells and the cells of the impulse connectingsystem such as those that constitute the sinoatrial node,atrioventricular node, and atrioventricular bundle. In one preferredembodiment striated muscle such as skeletal muscle or cardiac muscle istargeted by injecting the polynucleotide into the blood vessel supplyingthe tissue. In skeletal muscle an artery is the delivery vessel; incardiac muscle, an artery or vein is used.

[0023] Polymers

[0024] A polymer is a molecule built up by repetitive bonding togetherof smaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

[0025] One of our several methods of nucleic acid delivery to cells isthe use of nucleic acid-polycations complexes. It was shown thatcationic proteins like histones and protamines or synthetic polymerslike polylysine, polyarginine, polyomithine, DEAE dextran, polybrene,and polyethylenimine are effective intracellular delivery agents whilesmall polycations like spermine are ineffective.

[0026] A polycation is a polymer containing a net positive charge, forexample poly-L-lysine hydrobromide. The polycation can contain monomerunits that are charge positive, charge neutral, or charge negative,however, the net charge of the polymer must be positive. A polycationalso can mean a non-polymeric molecule that contains two or morepositive charges. A polyanion is a polymer containing a net negativecharge, for example polyglutamic acid. The polyanion can contain monomerunits that are charge negative, charge neutral, or charge positive,however, the net charge on the polymer must be negative. A polyanion canalso mean a non-polymeric molecule that contains two or more negativecharges. The term polyion includes polycation, polyanion, zwitterionicpolymers, and neutral polymers. The term zwitterionic refers to theproduct (salt) of the reaction between an acidic group and a basic groupthat are part of the same molecule. Salts are ionic compounds thatdissociate into cations and anions when dissolved in solution. Saltsincrease the ionic strength of a solution, and consequently decreaseinteractions between nucleic acids with other cations.

[0027] In one embodiment, polycations are mixed with polynucleotides forintravascular delivery to a cell. Polycations provide the advantage ofallowing attachment of DNA to the target cell surface. The polymer formsa cross-bridge between the polyanionic nucleic acids and the polyanionicsurfaces of the cells. As a result the main mechanism of DNAtranslocation to the intracellular space might be non-specificadsorptive endocytosis which may be more effective then liquidendocytosis or receptor-mediated endocytosis. Furthermore, polycationsare a very convenient linker for attaching specific receptors to DNA andas result, DNA-polycation complexes can be targeted to specific celltypes.

[0028] Additionally, polycations protect DNA in complexes againstnuclease degradation. This is important for both extra- andintracellular preservation of DNA. The endocytic step in theintracellular uptake of DNA-polycation complexes is suggested by resultsin which DNA expression is only obtained by incorporating a mildhypertonic lysis step (either glycerol or DMSO). Gene expression is alsoenabled or increased by preventing endosome acidification with NH₄CI orchloroquine. Polyethylenimine which facilitates gene expression withoutadditional treatments probably disrupts endosomal function itself.Disruption of endosomal function has also been accomplished by linkingthe polycation to endosomal-disruptive agents such as fusion peptides oradenoviruses.

[0029] Polycations also cause DNA condensation. The volume which one DNAmolecule occupies in complex with polycations is drastically lower thanthe volume of a free DNA molecule. The size of DNA/polymer complex maybe important for gene delivery in vivo. In terms of intravenousinjection, DNA needs to cross the endothelial barrier and reach theparenchymal cells of interest.

[0030] The average diameter of liver fenestrae (holes in the endothelialbarrier) is about 100 nm, increases in pressure and/or permeability canincrease the size of the fenestrae. The fenestrae size in other organsis usually less. The size of the DNA complexes is also important for thecellular uptake process. DNA complexes should be smaller than 200 nm inat least one dimension. After binding to the target cells theDNA-polycation complex is expected to be taken up by endocytosis.

[0031] Polymers may incorporate compounds that increase their utility.These groups can be incorporated into monomers prior to polymerformation or attached to the polymer after its formation. The genetransfer enhancing signal (Signal) is defined in this specification as amolecule that modifies the nucleic acid complex and can direct it to acell location (such as tissue cells) or location in a cell (such as thenucleus) either in culture or in a whole organism. By modifying thecellular or tissue location of the foreign gene, the expression of theforeign gene can be enhanced.

[0032] The gene transfer enhancing signal can be a protein, peptide,lipid, steroid, sugar, carbohydrate, nucleic acid or synthetic compound.The gene transfer enhancing signals enhance cellular binding toreceptors, cytoplasmic transport to the nucleus and nuclear entry orrelease from endosomes or other intracellular vesicles.

[0033] Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

[0034] Signals that enhance release from intracellular compartments(releasing signals) can cause DNA release from intracellularcompartments such as endosomes (early and late), lysosomes, phagosomes,vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network(TGN), and sarcoplasmic reticulum. Release includes movement out of anintracellular compartment into cytoplasm or into an organelle such asthe nucleus. Releasing signals include chemicals such as chloroquine,bafilomycin or Brefeldin A1 and the ER-retaining signal (KDEL sequence),viral components such as influenza virus hemagglutinin subunit HA-2peptides and other types of amphipathic peptides.

[0035] Cellular receptor signals are any signal that enhances theassociation of the gene with a cell. This can be accomplished by eitherincreasing the binding of the gene to the cell surface and/or itsassociation with an intracellular compartment, for example: ligands thatenhance endocytosis by enhancing binding the cell surface. This includesagents that target to the asialoglycoprotein receptor by usingasialoglycoproteins or galactose residues. Other proteins such asinsulin, EGF, or transferrin can be used for targeting. Peptides thatinclude the RGD sequence can be used to target many cells. Chemicalgroups that react with sulfhydryl or disulfide groups on cells can alsobe used to target many types of cells. Folate and other vitamins canalso be used for targeting. Other targeting groups include moleculesthat interact with membranes such as lipids fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives. In addition viralproteins could be used to bind cells.

[0036] Polynucleotides

[0037] The term nucleic acid is a term of art that refers to a string ofat least two base-sugar-phosphate combinations. (A polynucleotide isdistinguished from an oligonucleotide by containing more than 120monomeric units.) Nucleotides are the monomeric units of nucleic acidpolymers. The term includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA) in the form of an oligonucleotide messenger RNA, anti-sense,plasmid DNA, parts of a plasmid DNA or genetic material derived from avirus. Anti-sense is a polynucleotide that interferes with the functionof DNA and/or RNA. The term nucleic acids—refers to a string of at leasttwo base-sugar-phosphate combinations. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, but contain the same bases. Nucleotides are the monomericunits of nucleic acid polymers. The term includes deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). RNA may be in the form of an tRNA(transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA(messenger RNA), anti-sense RNA, and ribozymes. DNA may be in formplasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives ofthese groups. In addition these forms of DNA and RNA may be single,double, triple, or quadruple stranded. The term also includes PNAs(peptide nucleic acids), phosphorothioates, and other variants of thephosphate backbone of native nucleic acids.

[0038] A polynucleotide can be delivered to a cell to express anexogenous nucleotide sequence, to inhibit, eliminate, augment, or alterexpression of an endogenous nucleotide sequence, or to express aspecific physiological characteristic not naturally associated with thecell. Polynucleotides may be coded to express a whole or partialprotein, or may be anti-sense.

[0039] A delivered polynucleotide can stay within the cytoplasm ornucleus apart from the endogenous genetic material. Alternatively, thepolymer could recombine (become a part of) the endogenous geneticmaterial. For example, DNA can insert into chromosomal DNA by eitherhomologous or non-homologous recombination.

[0040] Vectors are polynucleic molecules originating from a virus, aplasmid, or the cell of a higher organism into which another nucleicfragment of appropriate size can be integrated without loss of thevectors capacity for self-replication; vectors typically introduceforeign DNA into host cells, where it can be reproduced. Examples areplasmids, cosmids, and yeast artificial chromosomes; vectors are oftenrecombinant molecules containing DNA sequences from several sources. Avector includes a viral vector: for example, adenovirus; DNA;adenoassociated viral vectors (AAV) which are derived fromadenoassociated viruses and are smaller than adenoviruses; andretrovirus (any virus in the family Retroviridae that has RNA as itsnucleic acid and uses the enzyme reverse transcriptase to copy itsgenome into the DNA of the host cell's chromosome; examples include VSVG and retroviruses that contain components of lentivirus including HIVtype viruses).

[0041] A non-viral vector is defined as a vector that is not assembledwithin an eukaryotic cell.

[0042] Permeability

[0043] In another preferred embodiment, the permeability of the vesselis increased. Efficiency of polynucleotide delivery and expression wasincreased by increasing the permeability of a blood vessel within thetarget tissue. Permeability is defined here as the propensity formacromolecules such as polynucleotides to move through vessel walls andenter the extravascular space. One measure of permeability is the rateat which macromolecules move through the vessel wall and out of thevessel. Another measure of permeability is the lack of force thatresists the movement of polynucleotides being delivered to leave theintravascular space.

[0044] To obstruct, in this specification, is to block or inhibit inflowor outflow of blood in a vessel. Rapid injection may be combined withobstructing the outflow to increase permeability. For example, anafferent vessel supplying an organ is rapidly injected and the efferentvessel draining the tissue is ligated transiently. The efferent vessel(also called the venous outflow or tract) draining outflow from thetissue is also partially or totally clamped for a period of timesufficient to allow delivery of a polynucleotide. In the reverse, anefferent is injected and an afferent vessel is occluded.

[0045] In another preferred embodiment, the intravascular pressure of ablood vessel is increased by increasing the osmotic pressure within theblood vessel. Typically, hypertonic solutions containing salts such asNaCl, sugars or polyols such as mannitol are used. Hypertonic means thatthe osmolarity of the injection solution is greater than physiologicosmolarity. Isotonic means that the osmolarity of the injection solutionis the same as the physiological osmolarity (the tonicity or osmoticpressure of the solution is similar to that of blood). Hypertonicsolutions have increased tonicity and osmotic pressure similar to theosmotic pressure of blood and cause cells to shrink.

[0046] In another preferred embodiment, the permeability of the bloodvessel can also be increased by a biologically-active molecule. Abiologically-active molecule is a protein or a simple chemical such aspapaverine or histamine that increases the permeability of the vessel bycausing a change in function, activity, or shape of cells within thevessel wall such as the endothelial or smooth muscle cells. Typically,biologically-active molecules interact with a specific receptor orenzyme or protein within the vascular cell to change the vessel'spermeability. Biologically-active molecules include vascularpermeability factor (VPF) which is also known as vascular endothelialgrowth factor (VEGF). Another type of biologically-active molecule canalso increase permeability by changing the extracellular connectivematerial. For example, an enzyme could digest the extracellular materialand increase the number and size of the holes of the connectivematerial.

[0047] In another embodiment a non-viral vector along with apolynucleotide is intravascularly injected in a large injection volume.The injection volume is dependent on the size of the animal to beinjected and can be from 1.0 to 3.0 ml or greater for small animals(i.e. tail vein injections into mice). The injection volume for rats canbe from 6 to 35 ml or greater. The injection volume for primates can be70 to 200 ml or greater. The injection volumes in terms of ml/bodyweight can be 0.03 ml/g to 0.1 ml/g or greater.

[0048] The injection volume can also be related to the target tissue.For example, delivery of a non-viral vector with a polynucleotide to alimb can be aided by injecting a volume greater than 5 ml per rat limbor greater than 70 ml for a primate. The injection volumes in terms ofml/limb muscle are usually within the range of 0.6 to 1.8 ml/g of musclebut can be greater. In another example, delivery of a polynucleotide toliver in mice can be aided by injecting the non-viralvector-polynucleotide in an injection volume from 0.6 to 1.8 ml/g ofliver or greater. In another preferred embodiment, delivering apolynucleotide-non-viral vector to a limb of a primate (rhesus monkey),the complex can be in an injection volume from 0.6 to 1.8 ml/g of limbmuscle or anywhere within this range.

[0049] In another embodiment the injection fluid is injected into avessel rapidly. The speed of the injection is partially dependent on thevolume to be injected, the size of the vessel to be injected into, andthe size of the animal. In one embodiment the total injection volume(1-3 mls) can be injected from 15 to 5 seconds into the vascular systemof mice. In another embodiment the total injection volume (6-35 mls) canbe injected into the vascular system of rats from 20 to 7 seconds. Inanother embodiment the total injection volume (80-200 mls) can beinjected into the vascular system of monkeys from 120 seconds or less.

[0050] In another embodiment a large injection volume is used and therate of injection is varied. Injection rates of less than 0.012 ml pergram (animal weight) per second are used in this embodiment. In anotherembodiment injection rates of less than ml per gram (target tissueweight) per second are used for gene delivery to target organs. Inanother embodiment injection rates of less than 0.06 ml per gram (targettissue weight) per second are used for gene delivery into limb muscleand other muscles of primates.

[0051] Reporter Molecules

[0052] There are three types of reporter (marker) gene products that areexpressed from reporter genes. The reporter gene/protein systemsinclude:

[0053] a) Intracellular gene products such as luciferase,β-galactosidase, or chloramphenicol acetyl transferase. Typically, theyare enzymes whose enzymatic activity can be easily measured.

[0054] b) Intracellular gene products such as β-galactosidase or greenfluorescent protein which identify cells expressing the reporter gene.On the basis of the intensity of cellular staining, these reporter geneproducts also yield qualitative information concerning the amount offoreign protein produced per cell.

[0055] c) Secreted gene products such as growth hormone, factor IX, oralpha1-antitrypsin are useful for determining the amount of a secretedprotein that a gene transfer procedure can produce. The reporter geneproduct can be assayed in a small amount of blood.

[0056] We have disclosed gene expression achieved from reporter genes inparenchymal cells. The terms “delivery,” “delivering geneticinformation,” “therapeutic” and “therapeutic results” are defined inthis application as representing levels of genetic products, includingreporter (marker) gene products, which indicate a reasonable expectationof genetic expression using similar compounds (nucleic acids), at levelsconsidered sufficient by a person having ordinary skill in the art ofdelivery and gene therapy. For example: Hemophilia A and B are caused bydeficiencies of the X-linked clotting factors VIII and IX, respectively.Their clinical course is greatly influenced by the percentage of normalserum levels of factor VIII or IX: <2%, severe; 2-5%, moderate; and5-30% mild. This indicates that in severe patients only 2% of the normallevel can be considered therapeutic. Levels greater than 6% preventspontaneous bleeds but not those secondary to surgery or injury. Aperson having ordinary skill in the art of gene therapy would reasonablyanticipate therapeutic levels of expression of a gene specific for adisease based upon sufficient levels of marker gene results. In theHemophilia example, if marker genes were expressed to yield a protein ata level comparable in volume to 2% of the normal level of factor VIII,it can be reasonably expected that the gene coding for factor VIII wouldalso be expressed at similar levels.

EXAMPLES Enhanced Delivery of Naked DNA

[0057] Enhancement of in Vivo Gene Expression by M-methyl-L-arginine(L-NMMA) Following Intravascular Delivery of Naked DNA:

[0058] Intravascular delivery of pCILuc via the iliac artery of ratfollowing a short pre-treatment with L-NMMA delivery enhancer. A 4 cmlong abdominal midline excision was performed in 150-200 g, adultSprague-Dawley rats anesthesized with 80 mg/mg ketamine and 40 mg/kgxylazine. Microvessel clips were placed on external iliac, caudalepigastric, internal iliac and deferent duct arteries and veins to blockboth outflow and inflow of the blood to the leg. 3 ml of normal salinewith 0.66 mM L-NMMA were injected into the external iliac artery. After2 min 27 g butterfly needle was inserted into the external iliac arteryand 10 ml of DNA solution (50 ug/ml pCILuc) in normal saline wasinjected within 8-9 sec. Luciferase assays was performed 2 days afterinjection on limb muscle samples (quadriceps femoris).

[0059] Organ Treatment Total Luciferase (Nanograms)

[0060] Muscle (quadriceps) +papaverine 9,999

[0061] Muscle (quadriceps) +0.66 mM L-NMMA 15,398

[0062] Muscle (quadriceps) +papaverine, +0.66 mM L-NMMA 24,829

[0063] 2) Enhancement of in Vivo Gene Expression by AurintricarboxylicAcid (ATA) Delivery Enhancer Following Intravascular Delivery of NakedDNA.

[0064] Intravascular delivery of pCILuc in the absence or presence ofaurintricarboxylic acid via tail vein injection into mice. 10 microgramsof pCILuc was diluted to 2.5 ml with Ringers solution andaurintricarboxylic acid was added to a final concentration of 0.11mg/ml. The DNA solution was injected into the tail vein of 25 g ICR micewith an injection time of ˜7 seconds. Mice were sacrificed 24 hoursafter injection and various organs were assayed for luciferaseexpression.

[0065] Organ Treatment Total Relative Light Units per Organ

[0066] Liver none 55,300,000,000

[0067] Liver +ATA 109,000,000,000

[0068] Spleen none 63,200,000

[0069] Spleen +ATA 220,000,000

[0070] Lung none 100,000,000

[0071] Lung +ATA 128,000,000

[0072] Heart none 36,700,000

[0073] Heart +ATA 32,500,000

[0074] Kidney none 15,800,000

[0075] Kidney +ATA 82,400,000

DNA/Polymer Delivery

[0076] Rapid injection of pDNA/cationic polymer complexes (containing 10μg of pCILuc; a luciferase expression vector utilizing the human CMVpromoter) in 2.5 ml of Ringers solution (147 mM NaCl, 4 mM KCl, 1.13 mMCaCl2) into the tail vein of ICR mice facilitated expression levelshigher than comparable injections using naked plasmid DNA (pCILuc).Maximal luciferase expression using the tail vein approach was achievedwhen the DNA solution was injected within 7 seconds. Luciferaseexpression was also critically dependent on the total injection volumeand high level gene expression in mice was obtained following tail veininjection of polynucleotide/polymer complexes of 1, 1.5, 2, 2.5, and 3ml total volume. There is a positive correlation between injectionvolume and gene expression for total injection volumes over 1 ml. Forthe highest expression efficiencies an injection delivery rate ofgreater than 0.003 ml per gram (animal weight) per second is likelyrequired. Injection rates of 0.004, 0.006, 0.009, 0.012 ml per gram(animal weight) per second yield successively greater gene expressionlevels.

[0077] The graph above illustrates high level luciferase expression inliver following tail vein injections of naked plasmid DNA and plasmidDNA complexed with labile disulfide containing polycationsL-cystine—1,4-bis(3-aminopropyl)piperazine copolymer (M66) and5,5′-Dithiobis(2-nitrobenzoic acid)—Pentaethylenehexamine Copolymer(M72). The labile polycations were complexed with DNA at a 3:1 wt:wtratio resulting in a positively charged complex. Complexes were injectedinto 25 gram ICR mice in a total volume of 2.5 ml of ringers solution.

[0078] The graph above indicates high level luciferase expression inspleen, lung, heart and kidney following tail vein injections of nakedplasmid DNA and plasmid DNA complexed with labile disulfide containingpolycations M66 and M72. The labile polycations were complexed with DNAat a 3:1 wt:wt ratio resulting in a positively charged complex.Complexes were injected into 25 gram ICR mice in a total volume of 2.5ml of ringers solution.

[0079] Luciferase Expression in a Variety of Tissues Following a SingleTail Vein Injection of pCILuc/66 Complexes:

[0080] DNA and polymer 66 were mixed at a 1:1.7 wt:wt ratio in water anddiluted to 2.5 ml with Ringers solution as described. Complexes wereinjected into tail vein of 25 g ICR mice within 7 seconds. Mice weresacrificed 24 hours after injection and various organs were assayed forluciferase expression.

[0081] Organ Total Relative Light Units

[0082] Prostate 637,000

[0083] Skin (abdominal wall) 194,000

[0084] Testis 589,000

[0085] Skeletal Muscle (quadriceps) 35,000

[0086] fat (peritoneal cavity) 44,700

[0087] bladder 17,000

[0088] brain 247,000

[0089] pancreas 2,520,000

[0090] Directed Intravascular Injection of pCILuc/66 Polymer Complexesinto Dorsal Vein of Penis Results in High Level Gene Rxpression in theProstate and Other Localized Tissues:

[0091] Complexes were formed as described for example above and injectedrapidly into the dorsal vein of the penis (within 7 seconds). Fordirected delivery to the prostate with increased hydrostatic pressure,clamps were applied to the inferior vena cava and the anastomotic veinsjust prior to the injection and removed just after the injection (within5-10 seconds). Mice were sacrificed 24 hours after injection and variousorgans were assayed for luciferase expression.

[0092] Organ Total Relative Light Units Per Organ

[0093] Prostate 129,982,450

[0094] Testis 4,229,000

[0095] fat (around bladder) 730,300

[0096] bladder 618,000

[0097] Intravascular Tail Vein Injection into Rat Results in High LevelGene Expression in a Variety of Organs:

[0098] 100 micrograms of pCILuc was diluted into 30 mls Ringers solutionand injected into the tail vein of 480 gram Harlan Sprague Dawley rat.The entire volume was delivered within 15 seconds. 24 hours afterinjection various organs were harvested and assayed for luciferaseexpression.

[0099] Organ Total Relative Light Units Per Organ

[0100] Liver 30,200,000,000

[0101] Spleen 14,800,000

[0102] Lung 23,600,000

[0103] Heart 5,540,000

[0104] Kidney 19,700,000

[0105] Prostate 3,490,000

[0106] Skeletal Muscle (quadriceps) 7,670,000

Cleavable Polymers

[0107] A prerequisite for gene expression is that once DNA/cationicpolymer complexes have entered a cell the polynucleotide must be able todissociate from the cationic polymer. This may occur within cytoplasmicvesicles (i.e. endosomes), in the cytoplasm, or the nucleus. We havedeveloped bulk polymers prepared from disulfide bond containingco-monomers and cationic co-monomers to better facilitate this process.These polymers have been shown to condense polynucleotides, and torelease the nucleotides after reduction of the disulfide bond. Thesepolymers can be used to effectively complex with DNA and can alsoprotect DNA from DNases during intravascular delivery to the liver andother organs. After internalization into the cells the polymers arereduced to monomers, effectively releasing the DNA, as a result of thestronger reducing conditions (glutathione) found in the cell. Negativelycharged polymers can be fashioned in a similar manner, allowing thecondensed nucleic acid particle (DNA+polycation) to be “recharged” witha cleavable anionic polymer resulting in a particle with a net negativecharge that after reduction of disulfide bonds will release thepolynucleic acid. The reduction potential of the disulfide bond in thereducible co-monomer can be adjusted by chemically altering thedisulfide bonds environment. This will allow the construction ofparticles whose release characteristics can be tailored so that thepolynucleic acid is released at the proper point in the deliveryprocess.

[0108] Cleavable Cationic Polymers

[0109] Cationic cleavable polymers are designed such that thereducibility of disulfide bonds, the charge density of polymer, and thefunctionalization of the final polymer can all be controlled. Thedisulfide co-monomer can have reactive ends chosen from, but not limitedto the following: the disulfide compounds contain reactive groups thatcan undergo acylation or alkylation reactions. Such reactive groupsinclude isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimideesters, succinimide esters, sulfonyl chloride, aldehyde, epoxide,carbonate, imidoester, carboxylate, alkylphosphate, arylhalides (e.g.difluoro-dinitrobenzene) or succinic anhydride.

[0110] If functional group A (cationic co-monomer) is an amine then B(disulfide containing comonomer) can be (but not restricted to) anisothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide, sulfonylchloride, aldehyde (including formaldehyde and glutaraldehyde), epoxide,carbonate, imidoester, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or succinic anhyride. In other terms whenfunction A is an amine then function B can be acylating or alkylatingagent.

[0111] If functional group A is a sulfhydryl then functional group B canbe (but not restricted to) an iodoacetyl derivative, maleimide, vinylsulfone, aziridine derivative, acryloyl derivative, fluorobenzenederivatives, or disulfide derivative (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid{TNB} derivatives).

[0112] If functional group A is carboxylate then functional group B canbe (but not restricted to) a diazoacetate or an amine, alcohol, orsulfhydryl in which carbonyldiimidazole or carbodiimide is used.

[0113] If functional group A is an hydroxyl then functional group B canbe (but not restricted to) an epoxide, oxirane, or an carboxyl group inwhich carbonyldiimidazole or carbodiimide or N,N′-disuccinimidylcarbonate, or N-hydroxysuccinimidyl chloroformate is used.

[0114] If functional group A is an aldehyde or ketone then function Bcan be (but not restricted to) an hydrazine, hydrazide derivative, amine(to form a Schiff Base that may or may not be reduced by reducing agentssuch as NaCNBH₃).

[0115] The polymer is formed by simply mixing the cationic, anddisulfide-containing co-monomers under appropriate conditions forreaction. The resulting polymer may be purified by dialysis orsize-exclusion chromatography.

[0116] The reduction potential of the disulfide bond can be controlledin two ways. Either by altering the reduction potential of the disulfidebond in the disulfide-containing co-monomer, or by altering the chemicalenvironment of the disulfide bond in the bulk polymer through choice theof cationic co-monomer.

[0117] The reduction potential of the disulfide bond in the co-monomercan be controlled by synthesizing new cross-linking reagents. Dimethyl3,3′-dithiobispropionimidate (DTBP) is a commercially availabledisulfide containing crosslinker from Pierce Chemical Co. This disulfidebond is reduced by dithiothreitol (DTT), but is only slowly reduced, ifat all by biological reducing agents such as glutathione. More readilyreducible crosslinkers have been synthesized by Mirus. Thesecrosslinking reagents are based on aromatic disulfides such as5,5′-dithiobis(2-nitrobenzoic acid) and 2,2′-dithiosalicylic acid. Thearomatic rings activate the disulfide bond towards reduction throughdelocalization of the transient negative charge on the sulfur atomduring reduction. The nitro groups further activate the compound toreduction through electron withdrawal which also stabilizes theresulting negative charge (FIG. 2).

[0118] Cleavable Disulfide Containing Co-Monomers:

[0119] Activated Disulfide Crosslinkers

[0120] diimidate activated crosslinkers

[0121] diimidate activated crosslinker with additional positive charge

[0122] di-NHS ester activated crosslinker with additional positivecharge

[0123] di-NHS ester activated crosslinker with no additional positivecharge

[0124] The reduction potential can also be altered by proper choice ofcationic co-monomer. For example when DTBP is polymerized along withdiaminobutane the disulfide bond is reduced by DTT, but not glutathione.When ethylenediamine is polymerized with DTBP the disulfide bond is nowreduced by glutathione. This is apparently due to the proximity of thedisulfide bond to the amidine functionality in the bulk polymer.

[0125] The charge density of the bulk polymer can be controlled throughchoice of cationic monomer, or by incorporating positive charge into thedisulfide co-monomer. For example spermine a molecule containing 4 aminogroups spaced by 3-4-3 methylene groups could be used for the cationicmonomer. Because of the spacing of the amino groups they would all bearpositive charges in the bulk polymer with the exception of the endprimary amino groups that would be derivitized during thepolymerization. Another monomer that could be used isN,N′-bis(2-aminoethyl)-1,3-propediamine (AEPD) a molecule containing 4amino groups spaced by 2-3-2 methylene groups. In this molecule thespacing of the amines would lead to less positive charge atphysiological pH, however the molecule would exhibit pH sensitivity,that is bear different net positive charge, at different pH's. Amolecule such as tetraethylenepentamine could also be used as thecationic monomer, this molecule consists of 5 amino groups each spacedby two methylene units. This molecule would give the bulk polymer pHsensitivity, due to the spacing of the amino groups as well as chargedensity, due to the number and spacing of the amino groups. The chargedensity can also be affected by incorporating positive charge into thedisulfide containing monomer, or by using imidate groups as the reactiveportions of the disulfide containing monomer as imidates are transformedinto amidines upon reaction with amine which retain the positive charge.

[0126] The bulk polymer can be designed to allow furtherfunctionalization of the polymer by incorporating monomers withprotected primary amino groups. These protected primary amines can thenbe deprotected and used to attach other functionalities such as nuclearlocalizing signals, endosome disrupting peptides, cell-specific ligands,fluorescent marker molecules, as a site of attachment for furthercrosslinking of the polymer to itself once it has been complexed with apolynucleic acid, or as a site of attachment for a second anionic layerwhen a cleavable polymer/polynucleic acid particle is being recharged toan anionic particle. An example of such a molecule is3,3′-(N′,N″-tert-butoxycarbonyl)-N-(3′-trifluoroacetamidylpropane)-N-methyldipropylammoniumbromide (see experimental), this molecule would be incorporated byremoving the two BOC protecting groups, incorporating the deprotectedmonomer into the bulk polymer, followed by deprotection of thetrifluoroacetamide protecting group.

[0127] Cleavable Anionic Polymers

[0128] Cleavable anionic polymers can be designed in much the samemanner as the cationic polymers. Short, multi-valent oligopeptides ofglutamic or aspartic acid can be synthesized with the carboxy terminuscapped with ethylene diamine. This oligo can the be incorporated into abulk polymer as a co-monomer with any of the amine reactive disulfidecontaining crosslinkers mentioned previously. A preferred crosslinkerwould make use of NHS esters as the reactive group to avoid retention ofpositive charge as occurs with imidates. The cleavable anionic polymerscan be used to recharge positively charged particles of condensedpolynucleic acids.

Examples of Cleavable Polymers

[0129]

[0130] Co-ethylenediamine/DTBP cleavable cationic polymer

[0131] Co-diaminobutane/DTBP cleavable cationic polymer

Co-Glutamic Acid/Activated Disulfide Cleavable Anionic Polymer

[0132] The cleavable anionic polymers can have co-monomers incorporatedto allow attachment of cell-specific ligands, endosome disruptingpeptides, fluorescent marker molecules, as a site of attachment forfurther crosslinking of the polymer to itself once it has been complexedwith a polynucleic acid, or as a site of attachment for to the initialcationic layer. For example the carboxyl groups on a portion of theanionic co-monomer could be coupled to an aminoalcohol such as4-hydroxybutylamine. The resulting alcohol containing comonomer can beincorporated into the bulk polymer at any ratio. The alcoholfunctionalities can then be oxidized to aldehydes, which can be coupledto amine containing ligands etc. in the presence of sodiumcyanoborohydride via reductive amination.

[0133] Synthesis of Activated Disulfide Containing Co-Monomers

[0134] Synthesis of 5,5′-dithiobis(2-nitrobenzoate)propionitrile:5,5′-dithiobis(2-nitrobenzoic acid) [Ellman's reagent] (500 mg,1.26mmol) was dissolved in 4.0 ml dioxane. Dicylohexylcarbodiimide (540 mg,2.6 mmol) and 3-hydroxypropionitrile (240 μL, 188 mg, 2.60 mmol) wereadded. The reaction mixture was stirred overnight at room temperature.The urea precipitate was removed by centrifugation. The dioxane wasremoved on rotary evaporator. The residue was washed with saturatedbicarbonate, water, and brine; and dried over magnesium sulfate. Solventremoval yielded 696 mg yellow/orange foam. The residue was purifiedusing normal phase HPLC (Alltech econosil, 250×22 nm), flow rate=9.0ml/min, mobile phase=1% ethanol in chloroform, retention time=13 min.Removal of solvent afforded 233 mg (36.8%) product as a yellow oil. TLC(silica: 5% methanol in chloroform; rf=0.51). H¹ NMR ∂ 8.05 (d, 4 H),7.75 (m, 4H), 4.55 (t, 4H), 2.85 (t, 4H).

[0135] Synthesis of 5,5′-dithiobis(2-nitrobenzoic acid)dimethylpropionimidate [DTNBP]: (113.5 mg, 0.226 mmol) was dissolved in 500 μLanhydrous chloroform along with anhydrous methanol (20.0 μL, 0.494mmol). The flask was stoppered with a rubber septum, chilled to 0° C. onan ice bath, and HCl gas produced by mixing sulfuric acid and ammoniumchloride was bubbled through the solution for a period of 10 minutes.The flask was then tightly sealed with parafilm and placed in a −20° C.freezer for a period of 48 hours. During this time a yellow oil formed.The oil was washed thoroughly with chloroform and dried under vacuum toyield 137 mg (95.8%) product as a yellow foam.

[0136] 3,340 -(N′,N″-tert-butoxycarbonyl)-N-methyldipropylamine (1).3,340 -Diamino-N-methyldipropylamine (0.800 ml, 0.721 g, 5.0 mmol) wasdissolved in 5.0 ml 2.2 N sodium hydroxide (11 mmol). To the solutionwas added Boc anhydride (2.50 ml, 2.38 g, 10.9 mmol) with magneticstirring. The reaction mixture was allowed to stir at room temperatureovernight (approximately 18 hours). The reaction mixture was made basicby adding additional 2.2 N NaOH until all t-butyl carboxylic acid was insolution. The solution was then extracted into chloroform (2×20 ml). Thecombined chloroform extracts were washed 2×10 ml water and dried overmagnesium sulfate. Solvent removal yielded 1.01 g (61.7%) product as awhite solid: ¹H-NMR (CDCl₃) δ 5.35 (bs, 2H), 3.17 (dt, 4H), 2.37 (t,4H), 2.15 (s, 3H), 1.65 (tt, 4H), 1.45 (s, 18H).

[0137] 3,3′-(N′,N″-tert-butoxycarbonyl)-N-(340-trifluoroacetamidylpropane)-N-methyldipropylammonium bromide (13).Compound 1 (100.6 mg, 0.291 mmol) and compound 4 (76.8 mg, 0.328 mmol)were dissolved in 0.150 ml dimethylformamide. The reaction mixture wasincubated at 50° C. for 3 days. TLC (reverse phase; acetonitrile: 50 mMammonium acetate pH 4.0; 3:1) showed 1 major and 2 minor spots none ofwhich corresponded to starting material. Recrystalization attempts wereunsuccessful so product was precipitated from ethanol with etheryielding 165.5 mg (98.2%) product and minor impurities as a clear oil:¹H-NMR (CDCl₃) δ 9.12 (bs,1H), 5.65 (bs, 2H), 3.50 (m, 8H), 3.20 (m,4H), 3.15 (s, 3H), 2.20 (m, 2H), 2.00 (m, 4H), 1.45 (s, 18H).

[0138] Intravascular Injections of DNA/Polymer Complexes

[0139] Synthesis of N,N′-Bis(t-BOC)-L-cystine:

[0140] To a solution of L-cystine (1 gm, 4.2 mmol, Aldrich ChemicalCompany) in acetone (10 ml) and water (10 ml) was added2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (2.5 gm, 10 mmol,Aldrich Chemical Company) and triethylamine (1.4 ml, 10 mmol, AldrichChemical Company). The reaction was allowed to stir overnight at roomtemperature. The water and acetone was then by rotary evaporationresulting in a yellow solid. The diBOC compound was then isolated byflash chromatography on silica gel eluting with ethyl acetate 0.1%acetic acid.

[0141] Synthesis of L-cystine—1,4-bis(3-aminopropyl)piperazine copolymer(M66):

[0142] To a solution of N,N′-Bis(t-BOC)-L-cystine (85 mg, 0.15 mmol) inethyl acetate (20 ml) was added N,N′-dicyclohexylcarbodiimide (108 mg,0.5 mmol) and N-hyroxysuccinimide (60 mg, 0.5 mmol). After 2 hr, thesolution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (54 μL, 0.25 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h. The ethylacetate was then removed by rotary evaporation and the resulting solidwas dissolved in trifluoroacetic acid (9.5 ml), water (0.5 ml) andtriisopropylsilane (0.5 ml). After 2 h, the trifluoroacetic acid wasremoved by rotary evaporation and the aqueous solution was dialyzed in a15,000 MW cutoff tubing against water (2×2 l) for 24 h. The solution wasthen removed from dialysis tubing, filtered through 5 μM nylon syringefilter and then dried by lyophilization to yield 30 mg of polymer.

[0143] Injection of plasmid DNA(pCILuc)/L-cystine—1,4-bis(3-aminopropyl)piperazine copolymer (M66)complexes into the iliac artery of rats.

[0144] Complex formation—500 ug pDNA (500 ul) was mixed with M66copolymer at a 1:3 wt:wt ratio in 500 ul saline. Complexes were thendiluted in Ringers solution to total volume of 10 mls.

[0145] Injections—total volume of 10 mls was injected into the iliacartery of Sprague-Dawley rats (Harlan, Indianapolis, Ind.) inapproximately 10 seconds.

[0146] Expression—Animals were sacrificed after 1 week and individualmuscle groups were removed and assayed for luciferase expression.

[0147] Rat hind limb muscle groups.

[0148] 1) upper leg posterior—6.46×10⁸ total Relative Light Units (32 ngluciferase)

[0149] 2) upper leg anterior—3.58×10⁹ total Relative Light Units (183 ngluciferase)

[0150] 3) upper leg middle—2.63×10⁹ total Relative Light Units (134 ngluciferase)

[0151] 4) lower leg anterior—3.19×10⁹ total Relative Light Units (163 ngluciferase)

[0152] 5) lower leg anterior—1.97×10⁹ total Relative Light Units (101 ngluciferase)

[0153] These results indicate that high level gene expression in allmuscle groups of the leg was facilitated by intravascular delivery ofpCILuc/M66 complexes into rat iliac artery.

[0154] Synthesis of 5,5′-Dithiobis[succinimidyl(2-nitrobenzoate):

[0155] 5,5′-dithiobis(2-nitrobenzoic acid) (50.0 mg, 0.126 mmol, AldrichChemical Company) and N-hyroxysuccinimide (29.0 mg, 0.252 mmol, AldrichChemical Company) were taken up in 1.0 ml dichloromethane.Dicylohexylcarbodiimide (52.0 mg, 0.252 mmol) was added and the reactionmixture was stirred overnight at room temperature. After 16 hr, thereaction mixture was partitioned in EtOAc/H₂O. The organic layer waswashed 2×H₂O, 1×brine, dried (MgSO ₄) and concentrated under reducedpressure. The residue was taken up in CH₂Cl₂, filtered, and purified byflash column chromatography on silica gel (130×30 mm, EtOAc:CH₂Cl₂ 1:9eluent) to afford 42 mg (56%)5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] as a white solid. H¹ NMR(DMSO) ∂ 7.81-7.77 (d, 2H), 7.57-7.26 (m, 4H), 3.69 (s, 8 H).

[0156] Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)—Pentaethylenehexamine Copolymer (M72):

[0157] Pentaethylenehexamine (4.2 μL, 0.017 mmol, Aldrich ChemicalCompany) was taken up in 1.0 ml dichloromethane and HCl (1 ml, 1 M inEt₂O, Aldrich Chemical Company) was added Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 ml DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded dropwise. After 16 hr, the solution was cooled, diluted with 3 mlH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 hr. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.9 mg (58%) of5,5′-dithiobis(2-nitrobenzoic acid)—pentaethylenehexamine Copolymer.

[0158] Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)—Tetraethylenepentamine Copolymer (#M57):

[0159] Tetraethylenepentamine (3.2 μL, 0.017 mmol, Aldrich ChemicalCompany) was taken up in 1.0 ml dichloromethane and HCl (1 ml, 1 M inEt₂O, Aldrich Chemical Company) was added Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 ml DMF and 5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg,0.017 mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (15 μL, 0.085 mmol, Aldrich Chemical Company) wasadded dropwise. After 16 hr, the solution was cooled, diluted with 3 mlH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.8 mg (62%) of5,5′-dithiobis(2-nitrobenzoic acid)—tetraethylenepentamine copolymer.

[0160] Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoic acid)—TetraethylenepentamineCopolymer Complexes:

[0161] Complexes were prepared as follows:

[0162] Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then2.5 ml Ringers was added.

[0163] Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then5,5′-Dithiobis(2-nitrobenzoic acid)—Tetraethylenepentamine Copolymer(336 μg) was added followed by 2.5 ml Ringers.

[0164] High pressure (2.5 ml) tail vein injections of the complex wereperformed as previously described (Zhang, G., Budker, V., Wolff, J.“High Levels of Foreign Gene Expression in Hepatocytes from Tail VeinInjections of Naked Plasmid DNA”, Human Gene Therapy, July, 1999).Results reported are for liver expression, and are the average of twomice. Luciferase expression was determined as previously reported(Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani,A., and Felgner, P. L., 1990 “Direct gene transfer into mouse muscle invivo,” Science 247, 1465-8.) A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used.

[0165] Results: High pressure injections

[0166] Complex I: 25,200,000 Relative Light Units

[0167] Complex II: 21,000,000 Relative Light Units

[0168] Results indicate that pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoic acid)—tetraethylenepentaminecopolymer complexes are nearly equivalent to pCI Luc DNA itself in highpressure injections. This indicates that the pDNA is being released fromthe complex and is accessible for transcription.

[0169] Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)—Tetraethylenepentamine—Tris(2-aminoethyl)amine Copolymer (#M58):

[0170] Tetraethylenepentamine (2.3 μL, 0.012 mmol, Aldrich ChemicalCompany) and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol, AldrichChemical Company) were taken up in 0.5 ml methanol and HCl (1 ml, 1 M inEt₂O, Aldrich Chemical Company) was added. Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 ml DMF and 5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg,0.017 mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (15 μL, 0.085 mmol, Aldrich Chemical Company) wasadded dropwise. After 16 hr, the solution was cooled, diluted with 3 mlH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 6.9 mg (77%) of5,5′-dithiobis(2-nitrobenzoicacid)—tetraethylenepentamine—tris(2-aminoethyl)amine copolymer.

[0171] Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)—Tetraethylenepentamine-Tris(2-aminoethyl)amine CopolymerComplexes:

[0172] Complexes were prepared as follows:

[0173] Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then2.5 ml Ringers was added.

[0174] Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then5,5′-Dithiobis(2-nitrobenzoicacid)—Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer (324 μg)was added followed by 2.5 ml Ringers.

[0175] High pressure (2.5 ml) tail vein injections of the complex wereperformed as previously described. Results reported are for liverexpression, and are the average of two mice. Luciferase expression wasdetermined a previously shown.

[0176] Results: High pressure injections

[0177] Complex I: 25,200,000 Relative Light Units

[0178] Complex II: 37,200,000 Relative Light Units

[0179] Results indicate that pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)—tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer Complexesare more effective than pCI Luc DNA in high pressure injections. Thisindicates that the pDNA is being released from the complex and isaccessible for transcription.

[0180] Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)—N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer (#M59):

[0181] N,N′-Bis(2-aminoethyl)-1,3-propanediamine (2.8 μL, 0.017 mmol,Aldrich Chemical Company) was taken up in 1.0 ml dichloromethane and HCl(1 ml, 1 M in Et₂O, Aldrich Chemical Company) was added. Et₂O was addedand the resulting HCl salt was collected by filtration. The salt wastaken up in 1 ml DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)](10 mg, 0.017 mmol) was added. The resulting solution was heated to 80°C. and diisopropylethylamine (12 μL, 0.068 mmol, Aldrich ChemicalCompany) was added dropwise. After 16 hr, the solution was cooled,diluted with 3 ml H₂O, and dialyzed in 12,000-14,000 MW cutoff tubingagainst water (2×2 L) for 24 hr. The solution was then removed fromdialysis tubing and dried by lyophilization to yield 5.9 mg (66%) of5,5′-dithiobis(2-nitrobenzoicacid)-N,N′-bis(2-aminoethyl)-1,3-propanediamine Copolymer.

[0182] Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)—N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer Complexes:

[0183] Complexes were prepared as follows:

[0184] Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then2.5 ml Ringers was added.

[0185] Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then5,5′-Dithiobis(2-nitrobenzoicacid)—N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer (474 μg) wasadded followed by 2.5 ml Ringers.

[0186] High pressure tail vein injections of 2.5 ml of the complex wereperformed as previously described. Results reported are for liverexpression, and are the average of two mice. Luciferase expression wasdetermined as previously shown.

[0187] Results: High pressure injections

[0188] Complex I: 25,200,000 Relative Light Units

[0189] Complex II: 341,000 Relative Light Units

[0190] Results indicate that pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoic acid)—tetraethylenepentamineCopolymer Complexes are less effective than pCI Luc DNA in high pressureinjections. Although the complex was less effective, the luciferaseexpression indicates that the pDNA is being released from the complexand is accessible for transcription.

[0191] Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)—N,N′-Bis(2-aminoethyl)-1,3-propanediamine—Tris(2-aminoethyl)amineCopolymer (#M60):

[0192] N,N′-Bis(2-aminoethyl)-1,3-propanediamine (2.0 μL, 0.012 mmol,Aldrich Chemical Company) and tris(2-aminoethyl)amine (0.51 μL, 0.0034mmol, Aldrich Chemical Company) were taken up in 0.5 ml methanol and HCl(1 ml, 1 M in Et₂O, Aldrich Chemical Company) was added. Et₂O was addedand the resulting HCl salt was collected by filtration. The salt wastaken up in 1 ml DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)](10 mg, 0.017 mmol) was added. The resulting solution was heated to 80°C. and diisopropylethylamine (12 μL, 0.068 mmol, Aldrich ChemicalCompany) was added dropwise. After 16 hr, the solution was cooled,diluted with 3 ml H₂O, and dialyzed in 12,000-14,000 MW cutoff tubingagainst water (2×2 L) for 24 hr. The solution was then removed fromdialysis tubing and dried by lyophilization to yield 6.0 mg (70%) of5,5′-dithiobis(2-nitrobenzoicacid)—N,N′-bis(2-aminoethyl)-1,3-propanediamine—tris(2-aminoethyl)aminecopolymer.

[0193] Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)—N,N′-Bis(2-aminoethyl)-1,3-propanediamine—Tris(2-aminoethyl)amineCopolymer Complexes:

[0194] Complexes were prepared as follows:

[0195] Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then2.5 ml Ringers was added.

[0196] Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then5,5′-Dithiobis(2-nitrobenzoicacid)—N,N′-Bis(2-aminoethyl)-1,3-propanediamine—Tris(2-aminoethyl)amineCopolymer (474 μg) was added followed by 2.5 ml Ringers.

[0197] High pressure tail vein injections of 2.5 ml of the complex werepreformed as previously described. Results reported are for liverexpression, and are the average of two mice. Luciferase expression wasdetermined as previously shown.

[0198] Results: High pressure injections

[0199] Complex I: 25,200,000 Relative Light Units

[0200] Complex II: 1,440,000 Relative Light Units

[0201] Results indicate that pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)—N,N′-Bis(2-aminoethyl)-1,3-propanediamine—Tris(2-aminoethyl)amineCopolymer Complexes are less effective than pCI Luc DNA in high pressureinjections. Although the complex was less effective, the luciferaseexpression indicates that the pDNA is being released from the complexand is accessible for transcription.

[0202] Synthesis of guanidino-L-cystine,1,4-bis(3-aminopropyl)piperazine copolymer (#M67):

[0203] To a solution of cystine (1 gm, 4.2 mmol) in ammonium hydroxide(10 ml) in a screw-capped vial was added O-methylisourea hydrogensulfate (1.8 gm, 10 mmol). The vial was sealed and heated to 60° C. for16 h. The solution was then cooled and the ammonium hydroxide wasremoved by rotary evaporation. The solid was then dissolved in water (20ml), filtered through a cotton plug. The product was then isolated byion exchange chromatography using Bio-Rex 70 resin and eluting withhydrochloric acid (100 mM).

[0204] Synthesis of guanidino-L-cystine1,4-bis(3-aminopropyl)piperazinecopolymer:

[0205] To a solution of guanidino-L-cystine (64 mg, 0.2 mmol) in water(10 ml) was slowly added N,N′-dicyclohexylcarbodiimide (82 mg, 0.4 mmol)and N-hyroxysuccinimide (46 mg, 0.4 mmol) in dioxane (5 ml). After 16hr, the solution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (40 μL, 0.2 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h and then theaqueous solution was dialyzed in a 15,000 MW cutoff tubing against water(2×2 1) for 24 h. The solution was then removed from dialysis tubing,filtered through 5 μM nylon syringe filter and then dried bylyophilization to yield 5 mg of polymer.

[0206] Particle Size of pDNA-L-cystine—1,4-bis(3-aminopropyl)piperazinecopolymer and DNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazineCopolymer Complexes:

[0207] To a solution of pDNA (10 μg/ml) in 0.5 ml 25 mM HEPES buffer pH7.5 was added 10 μg/ml L-cystine—1,4-bis(3-aminopropyl)piperazinecopolymer or guanidino-L-cystine1,4-bis(3-aminopropyl)piperazinecopolymer. The size of the complexes between DNA and the polymers weremeasured. For both polymers, the size of the particles wereapproximately 60 nm.

[0208] Condensation of DNA withL-cystine—1,4-bis(3-aminopropyl)piperazine copolymer and Decondensationof DNA Upon Addition of Glutathione:

[0209] Fluorescein labeled DNA was used for the determination of DNAcondensation in complexes withL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer. pDNA was modifiedto a level of 1 fluorescein per 100 bases using Mirus' LabelITFluorescein kit. The fluorescence was determined using a fluorescencespectrophotometer (Shimadzu RF-1501 spectrofluorometer) at an excitationwavelength of 495 nm and an emission wavelength of 530 nm (Trubetskoy,V. S., Slattum, P. M., Hagstrom, J. E., Wolff, J. A., and Budker, V. G.,“Quantitative assessment of DNA condensation,” Anal Biochem 267, 309-13(1999), incorporated herein by reference).

[0210] The intensity of the fluorescence of the fluorescein-labeled DNA(10 μg/ml) in 0.5 ml of 25 mM HEPES buffer pH 7.5 was 300 units. Uponaddition of 10 Rg/ml of L-cystine—1,4-bis(3-aminopropyl)piperazinecopolymer, the intensity decreased to 100 units. To this DNA-polycationsample was added 1 mM glutathione and the intensity of the fluorescencewas measured. An increase in intensity was measured to the levelobserved for the DNA sample alone. The half life of this increase influorescence was 8 minutes.

[0211] The experiment indicates that DNA complexes withphysiologically-labile disulfide-containing polymers are cleavable inthe presence of the biological reductant glutathione.

[0212] Mouse Tail Vein Injection ofDNA-L-cystine—1,4-bis(3-aminopropyl)piperazine copolymer andDNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymerComplexes:

[0213] Plasmid delivery in the tail vein of ICR mice was performed aspreviously described. To pCILuc DNA (50 μg) in 2.5 ml H₂O was addedeither L-cystine—1,4-bis(3-aminopropyl)piperazine copolymer,guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer, orpoly-L-lysine (34,000 MW, Sigma Chemical Company) (50 μg). The sampleswere then injected into the tail vein of mice using a 30 gauge, 0.5 inchneedle. One day after injection, the animal was sacrificed, and aluciferase assay was conducted. Polycation ng/liver poly-L-lysine 6.2L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer 439guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer 487

[0214] The experiment indicates that DNA complexes with thephysiologically-labile disulfide-containing polymers are capable ofbeing broken, thereby allowing the luciferase gene to be expressed.

[0215] Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)—Pentaethylenehexamine Copolymer (#M69):

[0216] Pentaethylenehexamine (4.2 μL, 0.017 mmol, Aldrich ChemicalCompany) was taken up in 1.0 ml dichloromethane and HCl (1 ml, 1 M inEt₂O, Aldrich Chemical Company) was added Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 ml DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded dropwise. After 16 hr, the solution was cooled, diluted with 3 mlH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 hr. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.9 mg (58%) of5,5′-dithiobis(2-nitrobenzoic acid)—pentaethylenehexamine Copolymer.

[0217] Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)—Pentaethylenehexamine—Tris(2-aminoethyl)amine Copolymer (#M70):

[0218] Pentaethylenehexamine (2.9 μL, 0.012 mmol, Aldrich ChemicalCompany) and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol, AldrichChemical Company) were taken up in 0.5 ml methanol and HCl (1 ml, 1 M inEt₂O, Aldrich Chemical Company) was added. Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 ml DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded dropwise. After 16 hr, the solution was cooled, diluted with 3 mlH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 6.0 mg (64%) of5,5′-dithiobis(2-nitrobenzoicacid)—pentaethylenehexamine—tris(2-aminoethyl)amine copolymer.

pH Cleavable Polymers for Intracellular Compartment Release

[0219] A cellular transport step that has importance for gene transferand drug delivery is that of release from intracellular compartmentssuch as endosomes (early and late), lysosomes, phagosomes, vesicle,endoplasmic reticulum, golgi apparatus, trans golgi network (TGN), andsarcoplasmic reticulum. Release includes movement out of anintracellular compartment into cytoplasm or into an organelle such asthe nucleus. Chemicals such as chloroquine, bafilomycin or Brefeldin A1.Chloroquine decreases the acidification of the endosomal and lysosomalcompartments but also affects other cellular functions. Brefeldin A, anisoprenoid fungal metabolite, collapses reversibly the Golgi apparatusinto the endoplasmic reticulum and the early endosomal compartment intothe trans-Golgi network (TGN) to form tubules. Bafilomycin A₁, amacrolide antibiotic is a more specific inhibitor of endosomalacidification and vacuolar type H⁺-ATPase than chloroquine. TheER-retaining signal (KDEL sequence) has been proposed to enhancedelivery to the endoplasmic reticulum and prevent delivery to lysosomes.

[0220] To increase the stability of DNA particles in serum, we haveadded to positively-charged DNA-polycation particles polyanions thatform a third layer in the DNA complex and make the particle negativelycharged. To assist in the disruption of the DNA complexes, we havesynthesized polymers that are cleaved in the acid conditions found inthe endosome, pH 5-7. We also have reason to believe that cleavage ofpolymers in the DNA complexes in the endosome assists in endosomedisruption and release of DNA into the cytoplasm.

[0221] There are two ways to cleave a polyion: cleavage of the polymerbackbone resulting in smaller polyions or cleavage of the link betweenthe polymer backbone and the ion resulting in an ion and an polymer. Ineither case, the interaction between the polyion and DNA is broken andthe number of molecules in the endosome increases. This causes anosomotic shock to the endosomes and disrupts the endosomes. In thesecond case, if the polymer backbone is hydrophobic it may interact withthe membrane of the endosome. Either effect may disrupt the endosome andthereby assist in release of DNA.

[0222] To construct cleavable polymers, one may attach the ions orpolyions together with bonds that are inherently labile such asdisulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,acetals, ketals, enol ethers, enol esters, imines, imminiums, andenamines. Another approach is construct the polymer in such a way as toput reactive groups, i.e. electrophiles and nucleophiles, in closeproximity so that reaction between the function groups is rapid.Examples include having carboxylic acid derivatives (acids, esters,amides) and alcohols, thiols, carboxylic acids or amines in the samemolecule reacting together to make esters, thiol esters, acid anhydridesor amides.

[0223] In one embodiment, ester acids and amide acids that are labile inacidic environments (pH less than 7, greater than 4) to form an alcoholand amine and an anhydride are use in a variety of molecules andpolymers that include peptides, lipids, and liposomes.

[0224] In one embodiment, ketals that are labile in acidic environments(pH less than 7, greater than 4) to form a diol and a ketone are use ina variety of molecules and polymers that include peptides, lipids, andliposomes.

[0225] In one embodiment, acetals that are labile in acidic environments(pH less than 7, greater than 4) to form a diol and an aldehyde are usein a variety of molecules and polymers that include peptides, lipids,and liposomes.

[0226] In one embodiment, enols that are labile in acidic environments(pH less than 7, greater than 4) to form a ketone and an alcohol are usein a variety of molecules and polymers that include peptides, lipids,and liposomes.

[0227] In one embodiment, iminiums that are labile in acidicenvironments (pH less than 7, greater than 4) to form an amine and analdehyde or a ketone are use in a variety of molecules and polymers thatinclude peptides, lipids, and liposomes.

[0228] pH-Sensitive Cleavage of Peptides and Polypeptides

[0229] In one embodiment, peptides and polypeptides (both referred to aspeptides) are modified by an anhydride. The amine (lysine), alcohol(serine, threonine, tyrosine), and thiol (cysteine) groups of thepeptides are modified by the an anhydride to produce an amide, ester orthioester acid. In the acidic environment of the internal vesicles (pHless than 6.5, greater than 4.5) (early endosomes, late endosomes, orlysosome) the amide, ester, or thioester is cleaved displaying theoriginal amine, alcohol, or thiol group and the anhydride.

[0230] A variety of endosomolytic and amphipathic peptides can be usedin this embodiment. A positively-charged amphipathic/endosomolyticpeptide is converted to a negatively-charged peptide by reaction withthe anhydrides to form the amide acids and this compound is thencomplexed with a polycation-condensed nucleic acid. After entry into theendosomes, the amide acid is cleaved and the peptide becomes positivelycharged and is no longer complexed with the polycation-condensed nucleicacid and becomes amphipathic and endosomolytic. In one embodiment thepeptides contains tyrosines and lysines. In yet another embodiment, thehydrophobic part of the peptide (after cleavage of the ester acid) is atone end of the peptide and the hydrophilic part (e.g. negatively chargedafter cleavage) is at another end. The hydrophobic part could bemodified with a dimethylmaleic anhydride and the hydrophilic part couldbe modified with a citranconyl anhydride. Since the dimethylmaleyl groupis cleaved more rapidly than the citrconyl group, the hydrophobic partforms first. In another embodiment the hydrophilic part forms alphahelixes or coil-coil structures.

[0231] pH-Sensitive Cleavage of Lipids and Liposomes

[0232] In another embodiment, the ester, amide or thioester acid iscomplexed with lipids and liposomes so that in acidic environments thelipids are modified and the liposome becomes disrupted, fusogenic orendosomolytic. The lipid diacylglycerol is reacted with an anhydride toform an ester acid. After acidification in an intracellular vesicle thediacylglycerol reforms and is very lipid bilayer disruptive andfusogenic.

[0233] Synthesis of Citraconylpolyvinylphenol

[0234] Polyvinylphenol (10 mg 30,000 MW Aldrich Chemical ) was dissolvedin 1 ml anhydrous pyridine. To this solution was added citraconicanhydride (100 μL, 1 mmol) and the solution was allowed to react for 16hr. The solution was then dissolved in 5 ml of aqueous potassiumcarbonate (100 mM) and dialyzed three times against 2 L water that wasat pH8 with addition of potassium carbonate. The solution was thenconcentrated by lyophilization to 10 mg/ml of citraconylpolyvinylphenol.

[0235] Synthesis of citraconylpoly-L-tyrosine

[0236] Poly-L-tyrosine (10 mg, 40,000 MW Sigma Chemical) was dissolvedin 1 ml anhydrous pyridine. To this solution was added citraconicanhydride (100 μL, 1 mmol) and the solution was allowed to react for 16hr. The solution was then dissolved in 5 ml of aqueous potassiumcarbonate (100 mM) and dialyzed against 3×2 L water that was at pH8 withaddition of potassium carbonate. The solution was then concentrated bylyophilization to 10 mg/ml of citraconylpoly-L-tyrosine.

[0237] Synthesis of citraconylpoly-L-lysine

[0238] Poly-L-lysine (10 mg 34,000 MW Sigma Chemical) was dissolved in 1ml of aqueous potassium carbonate (100 mM). To this solution was addedcitraconic anhydride (100 μL, 1 mmol) and the solution was allowed toreact for 2 hr. The solution was then dissolved in 5 ml of aqueouspotassium carbonate (100 mM) and dialyzed against 3×2 L water that wasat pH8 with addition of potassium carbonate. The solution was thenconcentrated by lyophilization to 10 mg/ml of citraconylpoly-L-lysine.

[0239] Synthesis of dimethylmaleylpoly-L-lysine

[0240] Poly-L-lysine (10 mg 34,000 MW Sigma Chemical ) was dissolved in1 ml of aqueous potassium carbonate (100 mM). To this solution was added2,3-dimethylmaleic anhydride (100 mg, 1 mmol) and the solution wasallowed to react for 2 hr. The solution was then dissolved in 5 ml ofaqueous potassium carbonate (100 mM) and dialyzed against 3×2 L waterthat was at pH8 with addition of potassium carbonate. The solution wasthen concentrated by lyophilization to 10 mg/ml ofdimethylmaleylpoly-L-lysine.

[0241] Characterization of Particles Formed with Citraconylated andDimethylmaleylated Polymers

[0242] To a complex of DNA (20 μg/ml) and poly-L-lysine (40 μg/ml) in1.5 ml was added the various citraconylpolyvinylphenol andcitraconylpoly-L-lysine (150 μg/ml). The sizes of the particles formedwere measured to be 90-120 nm and the zeta potentials of the particleswere measured to be −10 to −30 mV (Brookhaven ZetaPlus Particle Sizer).

[0243] To each sample was added acetic acid to make the pH 5. The sizeof the particles was measured as a function of time. Bothcitraconylpolyvinylphenol and citraconylpoly-L-lysine DNA complexes wereunstable under acid pH. The citraconylpolyvinylphenol sample hadparticles >1 μm in 5 minutes and citraconylpoly-L-lysine sample hadparticles >1 μm in 30 minutes.

[0244] Synthesis of Glutaric Dialdehyde—Poly-Glutamic acid (8mer)Copolymer

[0245] H₂N-EEEEEEEE-NHCH₂CH₂NH₂ (5.5 mg, 0.0057 mmol, Genosys) was takenup in 0.4 ml H₂O. Glutaric dialdehyde (0.52 μL, 0.0057 mmol, AldrichChemical Company) was added and the mixture was stirred at roomtemperature. After 10 min the solution was heated to 70° C. After 15hrs, the solution was cooled to room temperature and dialyzed againstH₂O (2×2L, 3500 MWCO). Lyophilization afforded 4.3 mg (73%) glutaricdialdehyde-poly-glutamic acid (8 mer) copolymer.

[0246] Synthesis of Ketal from Polyvinylphenyl Ketone and Glycerol

[0247] Polyvinyl phenyl ketone (500 mg, 3.78 mmol, Aldrich ChemicalCompany) was taken up in 20 ml dichloromethane. Glycerol (304 μL, 4.16mmol, Acros Chemical Company) was added followed by p-toluenesulfonicacid monohydrate (108 mg, 0.57 mmol, Aldrich Chemical Company). Dioxane(10 ml) was added and the solution was stirred at room temperatureovernight. After 16 hrs, TLC indicated the presence of ketone. Thesolution was concentrated under reduced pressure, and the residueredissolved in DMF (7 ml). The solution was heated to 60° C. for 16 hrs.Dialysis against H₂O (1×3L, 3500 MWCO), followed by Lyophilizationresulted in 606 mg (78%) of the ketal.

[0248] Synthesis of Ketal Acid of Polyvinylphenyl Ketone and GlycerolKetal

[0249] The ketal from polyvinylphenyl ketone and glycerol (220 mg, 1.07mmol) was taken up in dichloromethane (5 ml). Succinic anhydride (161mg, 1.6 mmol, Sigma Chemical Company) was added followed bydiisopropylethyl amine (0.37 ml, 2.1 mmol, Aldrich Chemical Company) andthe solution was heated at reflux. After 16 hrs, the solution wasconcentrated, dialyzed against H₂O (1×3L, 3500 MWCO), and lyophilized toafford 250 mg (75%) of the ketal acid.

[0250] Particle Sizing and Acid Lability of Poly-L-Lysine/Ketal Acid ofPolyvinylphenyl Ketone and Glycerol Ketal Complexes

[0251] Particle sizing (Brookhaven Instruments Corporation, ZetaPlusParticle Sizer, I90, 532 nm) indicated an effective diameter of 172 nm(40 μg) for the ketal acid Addition of acetic acid to a pH of 5 followedby particle sizing indicated a increase in particle size to 84000.

[0252] A poly-L-lysine/ketal acid (40 μg, 1:3 charge ratio) sampleindicated a particle size of 142 nm. Addition of acetic acid (5 μL, 6 N)followed by mixing and particle sizing indicated an effective diameterof 1970 nm. This solution was heated at 40° C. particle sizing indicateda effective diameter of 74000 and a decrease in particle counts.

[0253] Results:

[0254] The particle sizer data indicates the loss of particles upon theaddition of acetic acid to the mixture.

[0255] Synthesis of Ketal from Polyvinyl Alcohol and 4-AcetylbutyricAcid

[0256] Polyvinylalcohol (200 mg, 4.54 mmol, 30,000-60,000 MW, AldrichChemical Company) was taken up in dioxane (10 ml). 4-acetylbutyric acid(271 μL, 2.27 mmol, Aldrich Chemical Company) was added followed byp-toluenesulfonic acid monohydrate (86 mg, 0.45 mmol, Aldrich ChemicalCompany). After 16 hrs, TLC indicated the presence of ketone. Thesolution was concentrated under reduced pressure, and the residueredissolved in DMF (7 ml). The solution was heated to 60° C. for 16 hrs.Dialysis against H₂O (1×4L, 3500 MWCO), followed by lyophilizationresulted in 145 mg (32%) of the ketal.

[0257] Particle Sizing and Acid Lability of Poly-L-Lysine/Ketal fromPolyvinyl Alcohol and 4-Acetylbutyric Acid Complexes

[0258] Particle sizing (Brookhaven Instruments Corporation, ZetaPlusParticle Sizer, I90, 532 nm) indicated an effective diameter of 280 nm(743 kcps) for poly-L-lysine/ketal from polyvinyl alcohol and4-acetylbutyric acid complexes (1:3 charge ratio). A poly-L-lysinesample indicated no particle formation. Similarly, a ketal frompolyvinyl alcohol and 4-acetylbutyric acid sample indicated no particleformation. Acetic acid was added to the poly-L-lysine/ketal frompolyvinyl alcohol and 4-acetylbutyric acid complexes to a pH of 4.5.Particle sizing indicated particles of 100 nm, but at a minimal countrate (9.2 kcps)

[0259] Results:

[0260] The particle sizer data indicates the loss of particles upon theaddition of acetic acid to the mixture.

[0261] Synthesis of 1,4-Bis(3-aminopropyl)piperazine Glutaric DialdehydeCopolymer 1,4-Bis(3-aminopropyl)piperazine (206 μL, 0.998 mmol, AldrichChemical Company) was taken up in 5.0 ml H₂O . Glutaric dialdehyde was(206 μL, 0.998 mmol, Aldrich Chemical Company) was added and thesolution was stirred at room temperature. After 30 min, an additionalportion of H₂O was added (20 ml), and the mixture neutralized with 6 NHCl to pH 7, resulting in a red solution. Dialysis against H₂O (3×3L,12,000-14,000 MW cutoff tubing) and lyophilization afforded 38 mg (14%)of the copolymer

[0262] Particle Sizing and Acid Lability of pDNA (pCILuc)/1,4-Bis(3-aminopropyl)piperazine Glutaric Dialdehyde CopolymerComplexes (#M140)

[0263] To 50 μg pDNA in 2 ml HEPES (25 mM, pH 7.8) was added 135 μg1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymer. Particlesizing (Brookhaven Instruments Corporation, ZetaPlus Particle Sizer,I90, 532 nm) indicated an effective diameter of 110 nm for the complex.A 50 μg pDNA in 2 ml HEPES (25 mM, pH 7.8) sample indicated no particleformation. Similarly, a 135 μg 1,4-bis(3-aminopropyl)piperazine glutaricdialdehyde copolymer in 2 ml HEPES (25 mM, pH 7.8) sample indicated noparticle formation.

[0264] Acetic acid was added to the pDNA (pCILuc)/1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymercomplexes to a pH of 4.5. Particle sizing indicated particles of 2888nm, and aggregation was observed.

[0265] Results:

[0266] 1,4-Bis(3-aminopropyl)piperazine-glutaric dialdehyde copolymercondenses pDNA, forming small particles. Upon acidification, theparticle size increases, and aggregation occurs, indicating cleavage ofthe polymeric immine.

[0267] Mouse Tail Vein Injections of PDNA(pCILuc)/1,4-Bis(3-aminopropyl)piperazine Glutaric Dialdehyde CopolymerComplexes

[0268] Four complexes were prepared as follows:

[0269] Complex I: pDNA (pCI Luc, 50 μg) in 12.5 ml Ringers.

[0270] Complex II: pDNA (pCI Luc, 50 μg) was mixed with1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymer (50 μg)in 1.25 ml HEPES 25 mM, pH 8. This solution was then added to 11.25 mlRingers.

[0271] Complex III: pDNA (pCI Luc, 50 μg) was mixed with poly-L-lysine(94.5 μg, MW 42,000, Sigma Chemical Company) in 12.5 ml Ringers.

[0272] 2.5 ml tail vein injections of 2.5 ml of the complex werepreformed as previously described. Luciferase expression was determinedas previously indicated.

[0273] Results: 2.5 ml injections

[0274] Complex I: 3,692,000 Relative Light Units

[0275] Complex II: 1,047,000 Relative Light Units

[0276] Complex III: 4,379 Relative Light Units

[0277] Results indicate an increased level of pCI Luc DNA expression inpDNA /1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymercomplexes over pCI Luc DNA/poly-L-lysine complexes. These results alsoindicate that the pDNA is being released from the pDNA/1,4-Bis(3-aminopropyl)piperazine-glutaric dialdehyde copolymercomplexes, and is accessible for transcription.

Negatively Charged Complexes Using Non-cleavable polymers

[0278] Many cationic polymers such as histone (H1, H2a, H2b, H3, H4,H5), HMG proteins, poly-L-lysine, polyethylenimine, protamine, andpoly-histidine are used to compact polynucleic acids to help facilitategene delivery in vitro and in vivo. A key for efficient gene deliveryusing prior art methods is that the non-cleavable cationic polymers(both in vitro and in vivo) must be present in a charge excess over theDNA so that the overall net charge of the DNA/polycation complex ispositive. Conversely, using our intravascular delivery process havingnon-cleavable cationic polymer/DNA complexes we found that geneexpression is most efficient when the overall net charge of thecomplexes are negative (DNA negative charge>polyeation positive charge).Tail vein injections using cationic polymers commonly used for DNAcondensation and in vitro gene delivery revealed that high geneexpression occurred when the net charge of the complexes were negative.

[0279] Tail vein injection of pCILuc/polycation complexes in 2.5 mlringers solution into 25 gram mice (ICR, Harlan) as previously described(Zhang et al. Hum. Gen. Ther. 10:1735, 1999) Plasmid DNA encoding theluciferase gene was complexed with various polycations at two differentconcentrations. Complexes were prepared at polycation to DNA chargeratios of 0.5:1 (low) and 5:1 (high). This resulted in the formation ofnet negatively charged particles and net positively charged particlesrespectively. 24 hours after tail vein injection the livers wereremoved, cell extracts were prepared, and assayed for luciferaseactivity. Only complexes with a net negative overall charge displayedhigh gene expression following intravascular delivery.

[0280] The net surface charge of DNA/polymer particles formed at twodifferent polymer to DNA ratios was determined by zeta potentialanalysis. DNA/polymer complexes were formed by mixing the components atthe indicated charge: charge ratios in 25 mM HEPES, pH 8 at a DNAconcentration of 20 micrograms per ml (pCILuc). Complexes were assayedfor zeta potential on a Brookhaven ZetaPlus dynamic light scatteringparticle sizer/zeta potential analyzer.

[0281] Results:

[0282] DNA particles were formed at two different cationic polymer toDNA ratios of 0.5:1 (charge:charge) and 5:1 (charge:charge). At theseratios both negative (0.5:1 ratio) and positive particles (5:1 ratio)should be theoretically obtained. Zeta potential analysis of theseparticles confirmed that the two different ratios did yield oppositelycharged particles.

[0283] Cationic Polymer Cationic Polymer/DNA Ratio Zeta Potential (NetSurface Charge of Particle) Poly-L-lysine 0.5:1 − 16.77 mV Poly-L-lysine5:1 + 24.11 mV (n = 7) (n = 6) Polyethylenimine 0.5:1 − 12.47 mVPolyethylenimine 5:1 + 35.74 mV (n = 7) (n = 8) Histone H1 0.5:1 − 9.6mV (n = 8) Histone H1 5:1 + 20.97 mV (n = 8)

[0284]

[0285] High Efficiency Gene Expression Following Tail Vein Delivery ofpDNA/Cationic Peptide Complexes

[0286] Plasmid DNA (pCILuc) was mixed with an amphipathic cationicpeptide at a 1:2 ratio (charge ratio) and diluted into 2.5 ml of Ringerssolution per mouse. Complexes were injected into the tail vein of a 25 gICR mouse (Harlan Sprague Dawley, Indianapolis, Ind.) in 7 seconds.Animals were sacrificed after 24 hours and livers were removed andassayed for luciferase expression.

[0287] Complex Preparation (Per Mouse)

[0288] Complex I: pDNA (pCI Luc, 10 μg) in 2.5 ml Ringers.

[0289] Complex II: pDNA (pCI Luc, 10 μg) was mixed with cationic peptide(18 mer KLLKKLLKLWKKLLKKLK) at a 1:2 ratio. Complexes were diluted to2.5 ml with Ringers solution.

[0290] Tail vein injections of 2.5 ml of the complex were preformed aspreviously described. Luciferase expression was determined as previouslyshown.

[0291] Results: 2.5 ml injections

[0292] Complex I: 1.63×10¹⁰ Relative Light Units per liver

[0293] Complex II: 2.05×10¹⁰ Relative Light Units per liver

Negatively Charged Complexes Using Labile Polymers Delivery of PEI/DNAand Histone H1/DNA Particles to Rat Skeletal Muscle Via IntravascularInjection into an Artery.

[0294] Experimental Protocol and Methods:

[0295] PEI/DNA and histone H1/DNA particles were injected into rat legmuscle by either a single intra-arterial injection into the externaliliac [see Budker et al. Gene Therapy, 5:272, (1998)]. Harlan SpragueDawley (HSD SD) rats were used for the muscle injections. All rats usedwere female and approximately 150 grams and each received complexescontaining 100 micrograms of plasmid DNA encoding the luciferase geneunder control of the CMV enhancer/promoter (pCILuc) [see Zhang et al.Human Gene Therapy, 8:1763, (1997)].

[0296] Luciferase Assays:

[0297] Results of the rat injections are provided in relative lightunits (RLUs) and micrograms (μg) of luciferase produced. To determineRLUs, 10 μl of cell lysate were assayed using a EG&G Berthold LB9507luminometer and total muscle RLUs were determined by multiplying by theappropriate dilution factor. To determine the total amount of luciferaseexpressed per muscle we used a conversion equation that was determinedin an earlier study [see Zhang et al. Human Gene Therapy, 8:1763,(1997)] [pg luciferase=RLUs×5.1×10⁻⁵] Intravascular Delivery (IV Muscle)Total Total Muscle Group RLUs Luciferase DNA/PEI particles (1:0.5 chargeratio) muscle group 1 (upper leg anterior) 3.50 × 10⁹ 0.180 μg musclegroup 2 (upper leg posterior) 3.96 × 10⁹ 0.202 μg muscle group 3 (upperleg medial) 7.20 × 10⁹ 0.368 μg muscle group 4 (lower leg posterior)9.90 × 10⁹ 0.505 μg muscle group 5 (lower leg anterior) 9.47 × 10⁸ 0.048μg muscle group 6 (foot) 6.72 × 10⁶ 0.0003 μg  Total RLU/leg = 25.51 ×10⁹ RLU (1.303 μg luciferase) DNA/PEI particles (1:5 charge ratio)muscle group 1 (upper leg anterior) 1.77 × 10⁷ 0.0009 μg  muscle group 2(upper leg posterior) 1.47 × 10⁷ 0.0008 μg  muscle group 3 (upper legmedial) 5.60 × 10⁶ 0.00003 μg  muscle group 4 (lower leg posterior) 7.46× 10⁶ 0.00004 μg  muscle group 5 (lower leg anterior) 6.84 × 10⁶ 0.00003μg  muscle group 6 (foot) 1.55 × 10⁶ 0.000008 μg   Total RLU/leg = 5.39× 10⁷ RLU (0.0018 μg luciferase) DNA/histone H1 particles (1:0.5 chargeratio) muscle group 1 (upper leg anterior) 3.12 × 10⁹ 0.180 μg musclegroup 2 (upper leg posterior) 9.13 × 10⁹ 0.202 μg muscle group 3 (upperleg medial)  1.23 × 10¹⁰ 0.368 μg muscle group 4 (lower leg posterior)5.73 × 10⁹ 0.505 μg muscle group 5 (lower leg anterior) 4.81 × 10⁸ 0.048μg muscle group 6 (foot) 6.49 × 10⁶ 0.0003 μg  Total RLU/leg = 3.08 ×10¹⁰ RLU (1.57 μg luciferase) DNA/histone H1 particles (1:5 chargeratio) muscle group 1 (upper leg anterior) 1.42 × 10⁷ 0.0007 μg  musclegroup 2 (upper leg posterior) 5.94 × 10⁶ 0.0003 μg  muscle group 3(upper leg medial) 3.09 × 10⁶ 0.0002 μg  muscle group 4 (lower legposterior) 2.53 × 10⁶ 0.0001 μg  muscle group 5 (lower leg anterior)2.85 × 10⁶ 0.0001 μg  muscle group 6 (foot) 1.84 × 10⁵ 0.000009 μg  Total RLU/leg = 2.88 × 10⁷ RLU (0.0014 μg luciferase)

[0298] The foregoing is considered as illustrative only of theprinciples of the invention. Furthermore, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationshown and described. Therefore, all suitable modifications andequivalents fall within the scope of the invention.

1) A process for delivering a polynucleotide into an extravascularparenchymal cell of a mammal, comprising: a) inserting thepolynucleotide into a mammalian blood vessel, in vivo; b) increasing thepermeability of the blood vessel; c) passing the polynucleotide throughthe blood vessel into the extravascular space; d) delivering thepolynucleotide into the mammalian extravascular parenchymal cell; and,e) expressing the polynucleotide. 2) The process of claim 1 whereinincreasing the permeability of the blood vessel consists of increasingpressure against blood vessel walls. 3) The process of claim 2 whereinincreasing the pressure consists of increasing a volume of fluid withinthe blood vessel. 4) The process of claim 3 wherein increasing thevolume consists of inserting a solution containing the polynucleotideinto the blood vessel. 5) The process of claim 4 wherein increasedpressure is controlled by altering the volume of the solution inrelation to the time period of insertion. 6) The process of claim 5wherein the blood vessel consists of a tail vein. 7) The process ofclaim 1 wherein the cell is selected from the group consisting of aliver cell, spleen cell, heart cell, kidney cell, prostate cell, skincell, testis cell, skeletal muscle cell, fat cell, bladder cell, braincell, pancreas cell, thymus cell, and lung cell. 8) A process fordelivering a polynucleotide complexed with a compound into anextravascular parenchymal cell of a mammal, comprising: a) making apolynucleotide-compound complex wherein the zeta potential of thecomplex is less negative than the polynucleotide alone; b) addinganother compound to the complex to increase zeta potential negativity ofthe complex from the previous step; c) inserting the complex into amammalian blood vessel; d) increasing the permeability of the bloodvessel; e) passing the polynucleotide through the blood vessel; f)delivering the polynucleotide into the mammalian extravascularparenchymal cell; and, g) expressing the polynucleotide. 9) The processof claim 8 wherein increasing the permeability of the blood vesselconsists of increasing pressure against blood vessel walls. 10) Theprocess of claim 9 wherein increasing the pressure consists ofincreasing a volume of fluid within the blood vessel. 11) The process ofclaim 10 wherein increasing the volume consists of inserting a solutioncontaining the polynucleotide into the blood vessel. 12) The process ofclaim 11 wherein a specific volume of the solution is inserted within aspecific time period. 13) The process of claim 12 wherein increasedpressure is controlled by altering the volume of the solution inrelation to the time period of insertion. 14) The process of claim 13wherein the blood vessel consists of a tail vein. 15) The process ofclaim 8 wherein the cell is selected from the group consisting of aliver cell, spleen cell, heart cell, kidney cell, prostate cell, skincell, testis cell, skeletal muscle cell, fat cell, bladder cell, braincell, pancreas cell, thymus cell, and lung cell. 16) The process ofclaims 1 and 8 wherein the polynucleotide is inserted in at least a 1milliliter solution. 17) The process of claims 1 and 8 wherein theextravascular parenchymal space consists of the hepatocytes. 18) Theprocess of claim 17 wherein intrahepatic parenchymal pressure is atleast 10 mm mercury.